Frank Schmitz

The DNA sugar backbone 2' deoxyribose determines toll-like receptor 9 activation.

CpG motifs within phosphorothioate (PS)-modified DNA drive Toll-like receptor 9 (TLR9) activation, but the rules governing recognition of natural phosphodiester (PD) DNA are less understood. Here, we showed that the sugar backbone determined DNA recognition by TLR9. Homopolymeric, base-free PD 2 deoxyribose acted as a basal TLR9 agonist as it bound to and activated TLR9. This effect was enhanced by DNA bases, even short of CpG motifs. In contrast, PS-modified 2 deoxyribose homopolymers acted as TLR9 and TLR7 antagonists. They displayed high affinity to both TLRs and did not activate on their own, but they competitively inhibited ligand-TLR interaction and activation. Although addition of random DNA bases to the PS 2 deoxyribose backbone did not alter these effects, CpG motifs transformed TLR9-inhibitory to robust TLR9-stimulatory activity. Our results identified the PD 2 deoxyribose backbone as an important determinant of TLR9 activation by natural DNA, restrict CpG-motif dependency of TLR9 activation to synthetic PS-modified ligands, and define PS-modified 2 deoxyribose as a prime effector of TLR9 and TLR7 inhibition.

Endosomal Translocation of Vertebrate DNA Activates Dendritic Cells via TLR9-Dependent and -Independent Pathways

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.

Vaccination with Plasmid DNA Activates Dendritic Cells via Toll-Like Receptor 9 (TLR9) but Functions in TLR9-Deficient Mice

We analyzed whether the immunobiology of vaccinating plasmid DNA containing a transcription unit for OVA is influenced by immunostimulatory CpG motifs in the plasmid backbone. Indeed, plasmid DNA differentially activated in vitro myeloid and plasmacytoid dendritic cells (DCs) provided they expressed the CpG-DNA receptor, Toll-like receptor 9 (TLR9). Dependent on the DC subset, activation resulted in type 1 IFN production, while both DC subsets produced IL-6 and up-regulated expression of costimulatory molecules CD40 and CD86. In vivo, however, even upon repeated vaccination with plasmid DNA, priming of OVA-specific CTL and clonal expansion of SIINFEKL-specific CD8 T cells were equal in TLR9-positive and TLR9- or MyD88-negative mice. Overall, these results negate a dominant role of CpG-DNA/TLR9 interactions in long-term vaccination protocols.

Mammalian target of rapamycin (mTOR) orchestrates the defense program of innate immune cells.

The mammalian target of rapamycin (mTOR) can be viewed as cellular master complex scoring cellular vitality and stress. Whether mTOR controls also innate immune‐defenses is currently unknown. Here we demonstrate that TLR activate mTOR via phosphoinositide 3‐kinase/Akt. mTOR physically associates with the MyD88 scaffold protein to allow activation of interferon regulatory factor‐5 and interferon regulatory factor‐7, known as master transcription factors for pro‐inflammatory cytokine‐ and type I IFN‐genes. Unexpectedly, inactivation of mTOR did not prevent but increased lethality of endotoxin‐mediated shock, which correlated with increased levels of IL‐1β. Mechanistically, mTOR suppresses caspase‐1 activation, thus inhibits release of bioactive IL‐1β. We have identified mTOR as indispensable component of PRR signal pathways, which orchestrates the defense program of innate immune cells.

CpG-DNA aided cross-priming by cross-presenting B cells.

Covalent linkage of immunostimulatory CpG-DNA to OVA (CpG-OVA complex) results in CpG-DNA-aided cross-presentation of OVA by dendritic cells (DCs). In this study, we analyzed the thesis that CpG-OVA complexes may be cross-presented by B cells to route internalized Ag into the class I MHC presentation pathway. First, we describe that conjugation of CpG-DNA to OVA enhances up to 40-fold internalization of OVA by B cells, which in turn generate the CD8 T cell epitope SIINFEKL complexed to MHC class I, albeit less efficiently than DCs. Furthermore, upon internalization, CpG-DNA conjugated to OVA stimulates B cells to up-regulate costimulatory molecules and cytokines including IL-12. Adoptive transfer of CpG-OVA complex-loaded wild-type B cells cross-primes naive CD8 T cells both in wild-type mice and in MyD88-deficient mice. Overall, these findings disclose attributes of B cells, including cross-presentation of exogenous Ag and cross-priming of naive CD8 T cells that hitherto have been considered as hallmarks restricted to DCs.

Interferon‐regulatory‐factor 1 controls Toll‐like receptor 9‐mediated IFN‐β production in myeloid dendritic cells

Activation of interferon regulatory factor (IRF)‐3 and/or IRF‐7 drives the expression of antiviral genes and the production of α/β IFN, a hallmark of antiviral responses triggered by Toll‐like receptors (TLR). Here we describe a novel antiviral signaling pathway operating in myeloid (m) dendritic cells (DC) and macrophages that does not require IRF‐3 and/or IRF‐7 but is driven by IRF‐1. IRF‐1 together with myeloid differentiation factor 88 (MyD88) or IL‐1 receptor‐associated kinase (IRAK)‐1 triggered IFN‐β promoter activation. IRF‐1 physically interacted with MyD88 and activation of mDC via TLR‐9 induced IRF‐1‐dependent IFN‐β production paralleled by rapid transcriptional activation of IFN‐stimulated genes. The NF‐κB‐dependent production of pro‐inflammatory cytokines, however, was not influenced by IRF‐1. TLR‐9 signaling through this pathway conferred cellular antiviral resistance while IRF‐1‐deficient mice displayed enhanced susceptibility to viral infection. These results demonstrate that TLR‐9 activation of mDC and macrophages contributes to antiviral immunity via IRF‐1.

CpG motif‐independent activation of TLR9 upon endosomal translocation of “natural” phosphodiester DNA

Endosomally translocated host (self) DNA activates Toll‐like receptor 9 (TLR9), while extracellular self‐DNA does not. This inconsistency reflects poor endosomal DNA translocation but also implies that host DNA contains DNA sequences that function as ligands for TLR9. Herein we report that contrary to phosphorothioate (PS)‐stabilized oligonucleotides (ODN), “natural” phosphodiester (PD) ODN lacking CpG motifs activate TLR9. CpG motif‐independent TLR9 activation of Flt3‐L‐induced dendritic cells (DC) was dependent on enforced endosomal translocation and triggered upregulation of CD40 and CD69 as well as production of IL‐6 and IFN‐α. Binding studies utilizing surface plasmon resonance technology (Biacore) revealed low TLR9 binding to single‐stranded (ss) PD‐ODN lacking CpG motifs. At higher concentrations their TLR9 binding activity compared well with TLR9 binding of canonical ss PD CpG‐ODN. These results imply that both the chemical modification of the DNA backbone as well as the amount of endosomally translocated DNA represent determining factors that allow CpG motif‐independent activation of TLR9 by ss PD‐DNA.

Antigen co-encapsulated with adjuvants efficiently drive protective T cell immunity

Compared to live vaccines, the immunogenicity of subunit vaccines based on recombinant antigen (Ag) is poor, presumably because exogenous Ag fails to effectively access the endosomal Ag‐processing pathways of Ag‐presenting cells (APC). To overcome this limitation, we exploited biodegradable poly(lactic‐co‐glycolic) microspheres (MP) co‐entrapping Ag and Toll‐like receptor (TLR) 9 or 7 ligands as an endosomal delivery device. In vitro, microspheres were rapidly phagocytosed by APC and translocated into phago‐endosomal compartments, followed by degradation of the Ag and concurrent activation of endosomal TLR. As a consequence, full maturation of and cytokine secretion by APC as well as Ag‐cross‐presentation ensued. In vivo, loaded microspheres triggered clonal expansion of primary and secondary Ag‐specific CD4 and CD8 T cells. The efficacy of CD8 T cell cross‐priming was comparable to that of live vectors. The potency of T cell vaccination was demonstrated by protective and therapeutic interventions using infection‐ and tumor‐model systems. These preclinical subunit vaccination data thus recommend MP as a generally applicable and powerful endosomal delivery device of exogenous Ag plus TLR‐based adjuvants to vaccinate for protective and therapeutic CD4 and CD8 T cell immunity.

Control of dual-specificity phosphatase-1 expression in activated macrophages by IL-10

Ligation of Toll‐like receptors (TLR) on macrophages induces cytokines and mediators important for the control of pathogens. Macrophage activation has to be tightly controlled to prevent hyper‐inflammation. Accordingly, the hallmarks of TLR‐triggered signaling, nuclear translocation of NF‐κB and phosphorylation of mitogen‐activated protein kinases (MAPK), are transient events. We have mined microarray datasets for changes in the expression of phosphatases in resting and TLR‐activated macrophages. Several members of the dual‐specificity phosphatases (DUSP) were induced upon triggering TLR4 with LPS. Up‐regulation of DUSP1 mRNA was transient after stimulation with LPS alone, but addition of the immunosuppressive cytokine IL‐10 resulted in robust, continued DUSP1 expression. IL‐10 also synergized with the anti‐inflammatory glucocorticoid dexamethasone in the induction of DUSP1 mRNA expression in activated macrophages, as well as in the inhibition of IL‐6 and IL‐12 production. Increased expression of DUSP1 in IL‐10‐treated activated macrophages was correlated with a faster down‐regulation of p38 MAPK activation. Thus, these data suggest an operational link between IL‐10 and inibition of p38 MAPK via sustained expression of DUSP1.

Protective CD8 T Cell Immunity Triggered by CpG-Protein Conjugates Competes with the Efficacy of Live Vaccines

In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 μg of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.